The long-term objective of this research proposal is to develop anticancer drugs in the antifolate series that are more specific in their mode of action and less toxic to the host. To achieve this objective several classes of folate analogues will be synthesized and evaluated for their anticancer activity using the appropriate biochemical and pharmacological test systems. The proposed compounds are: 1) analogues of folic acid and homofolic acid that can be reduced by dihydrofolate reductase (DHFR) to 10- substituted tetrahydrofolates that are potentially capable of inhibiting other folate dependent enzymes; 2) specific inhibitors of thymidylate synthase (TS) that are analogues of 10-propargyl- 5,8-dideazafolate (PDDF) potentially capable of undergoing enhanced polyglutamylation; 3) non-classical analogues of PDDF without a quinazoline or pteridine ring system; 4) powerful inhibitors of DHFR and TS that may, a) either inhibit folylpolyglutamate synthetase (FPGS) or b) that may not function as substrates of FPGS and 5) potential inhibitors of GAR- transformylase. All these compounds will be evaluated for their: a) antitumor activity using folate requiring microorganisms; b) enzyme inhibitory potencies using both bacterial and mammalian enzymes in vitro; c) antitumor activity using tumor cells in culture; and d) antitumor activity in mice bearing methotrexate sensitive and methotrexate resistant L1210 leukemia. Those compounds which show promising preliminary activities in the above test systems will be further evaluated for their substrate or inhibitory activities towards dihydrofolate reductase and thymidylate synthase derived from human cell lines. In addition, they will be tested as inhibitors of the growth of several human cell lines in culture. (Human KB, KB/6B, Raji Burkitt lymphoma, Manca B-cell leukemia and MCF-7 cells.)